Characterization of Endofungal Bacteria and Their Role in the Ectomycorrhizal Fungus Helvella bachu

<i>Helvella bachu</i>, an ectomycorrhizal fungus, forms a symbiotic relationship with <i>Populus euphratica</i>, a rare and endangered species crucial to desert riparian ecosystems. In this study, endofungal bacteria (EFBs) within the fruiting bodies of <i>H. bachu</i> were confirmed by a polyphasic approach, including genomic sequencing, real-time quantitative PCR targeting the 16S rRNA gene, full-length and next-generation sequencing (NGS) of the 16S rRNA gene, and culture methods. The genera <i>Stenotrophomonas</i>, <i>Variovorax</i>, <i>Acidovorax</i>, and <i>Pedobacter</i> were abundant in the EFBs of fruiting bodies associated with three <i>Populus</i> hosts and were consistently present across different developmental stages. Notably, <i>S. maltophilia</i> and <i>V. paradoxus</i> were detected in high abundance, as revealed by full-length 16S rRNA sequencing, with <i>S. maltophilia</i> also isolated by culture methods. KO-pathway analysis indicated that pathways related to primary, secondary, and energy metabolism were predominantly enriched, suggesting these bacteria may promote <i>H. bachu</i> growth by producing essential compounds, including sugars, proteins, and vitamins, and secondary metabolites. This study confirmed the presence of EFBs in <i>H. bachu</i> and provided the first comprehensive overview of their structure, functional potential, and dynamic changes throughout fruiting body maturation, offering valuable insights for advancing the artificial domestication of this species.